Soluble secreted proteins that are expressed uniquely in specific organs, or proteins whose formation or secretion is regulated by disease states, are excellent markers for disease. The reason for this is that the disease can be diagnosed by simply measuring the level of the secreted protein in serum of a potential patient. The level of a secreted protein in serum can be easily measured in a number of different ways that are well known in the art, such as ELISA assay and Western blotting, directed at quantitating marker levels in the serum sample. However, in order to have a good marker for disease, the secreted protein must have distinctly different levels in normal and disease tissues. In order to provide accurate diagnosis in diseases that must be caught at early development stages in order to enable efficient treatment, such as cancer or fibrosis, the marker must have distinct expression or secretion levels even at an early stage of disease development.
Hepatoma, or hepatocellular carcinoma, is the most common primary liver cancer. In certain areas of the world, hepatomas are more common than metastatic liver cancer, and are a prominent cause of death.
Hepatocarcinoma can often arise as a complication of liver cirrhosis. Approximately 2-7% of patients with liver cirrhosis develop hepatocellular carcinoma, and a much higher percentage eventually need a liver transplant due to liver damage caused by the cirrhosis itself.
Liver function can be affected by many chemicals, medicines, diet regimes, environmental poisons, alcohol abuse and viral infections that lead to hepatitis. The most common complications are liver fibrosis and cirrhosis. Generally, the origins of liver fibrosis that leads in its advanced stages to cirrhosis are common complications of Hepatitis B and C.
Hepatitis B is very common in Africa and in Asia, especially in the Philippines and in China and is endemic in the Middle East. In Europe and North America the incidence of known carriers is about 1 in a 1000 people. Worldwide, it is estimated that there are over 350 million hepatitis B (HBV) carriers, which represents 5% of the world's population. In addition it is estimated that 10 to 30 million people are infected with the hepatitis B virus each year. 10% of the people infected with HBV develop chronic infection. People with a chronic HBV infection are at risk of liver damage and around 20-30% of these people later develop cirrhosis (http://hepatitis-central.com/).
Hepatitis C is almost as common, and it is estimated that there are approximately 200 million people worldwide infected with the virus. There are up to 230,000 new HCV infections every year in the U.S. alone. Currently, 8,000 to 10,000 people infected with HCV die each year. Over the next 10-20 years, chronic HCV is predicted to become a major burden on the health care system, as patients who are currently asymptomatic with a relatively mild form of the disease, progress to end-stage liver disease and develop hepatocellular carcinoma. Progressive hepatic fibrosis and cirrhosis develop in 20% to 30% of patients with chronic HCV. There is no vaccine and no completely effective treatment for this virus (http://hepatitis-central.com/). Predictions in the USA indicate that there will be a 60% increase in the incidence of cirrhosis, a 68% increase in hepatoma incidence, a 279% increment in incidence of hepatic decompensation, a 528% increase in the need for transplantation, and a 223% increase in liver death rate. Altogether the number of fibrotic and cirrhotic patients worldwide in need of periodic diagnosis can be estimated at around 20 million, with up to 2 million added each year. With regard to the number of pre-fibrotic patients that would benefit from an early diagnosis, there could be several hundred million worldwide.
Frequently the first symptoms of a hepatoma are abdominal pain, weight loss, and at a later stage of tumor development a large mass that can be felt in the upper right abdomen. However, as the initial symptoms are non-specific, they are often attributed to other possible conditions, and therefore biochemical and histological tests, which would give a more accurate diagnosis, are only performed when the tumor is largely developed.
Generally the survival rate for people in the United States with a hepatoma is poor because the tumor is normally detected at a late stage. In some other countries, such as Japan, the survival rate is higher because of routine screening and thus earlier detection.
Recurrence or development of second liver tumors is very common after therapy of hepatomas. Therefore, screening hepatoma patients with diagnostic methods is extremely important.
The gold standard for diagnosis of hepatomas is liver biopsy, but it cannot be performed on a routine basis, due to the invasiveness involved, and the complexity of the procedure.
Non-invasive serum markers may also be used for diagnosis, however in the case of liver cancer, the existing non-invasive serum markers are not satisfactory for the purpose of diagnosis, and even less suitable for early diagnosis of hepatomas.
The most established non-invasive tumor marker for hepatoma is α-fetoprotein that shows elevated levels following hepatocarcinogenesis. However, about 40% of the patients with small-sized hepatocarcinomas show normal α-fetoprotein levels (1).
As mentioned above, hepatocarcinoma can arise as a complication of liver cirrhosis. In the case of cirrhosis and fibrosis, early diagnosis is necessary to allow any potential treatment. Similar to the case of hepatomas, initial stages of liver fibrosis (e.g. arising from HBV or HCV infections) are asymptomatic or have mild symptoms often attributed to other possible conditions. Therefore, the disease is detected at a stage that is late for an effective treatment.
Generally, blood tests for liver function are based on the level of several markers such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma glutamic transpeptidase (GGT), bilirubin, albumin and prothrombin time (PT) in the serum. However, while the markers used in such “liver function tests” are capable of assessing hepatocyte integrity, which might be indicative to liver damage, most of them, except albumin and prothrombin, are not indicative of the synthesis function of the liver. Albumin, which is produced in the liver and circulates in the blood, is affected only when a liver disease is at a severe stage. On the other hand non-hepatic diseases such as nephrotic syndromes can affect albumin levels. Similarly, prothrombin, which is used to evaluate blood clotting disorders, is insensitive to mild liver disease and can be also affected by non-hepatic conditions such as dietary deficiencies or the use of anti-coagulants. Likewise, abnormal levels of bilirubin can result from hemolysis, ineffective erythropoiesis and other non-hepatic syndromes. In addition, as ALT, AST, ALP and GGT are also produced in organs other than the liver, their blood levels can be elevated in a wide range of non-hepatic diseases. Biochemical screening of healthy, asymptomatic people has revealed that up to 6% of the population exhibit abnormal levels of liver enzymes. However, the prevalence of liver disease in the general population is significantly lower (about 1%) (Gopal and Rosen, 2000). Even though the current serum biochemical test pattern may suggest a specific diagnosis, confirmation usually requires further investigation using imaging studies and, possibly, liver biopsy. Even mild liver test abnormalities may be an early clue to the presence of potentially significant liver disease (Hay, J. E., et al., 1989). For instance, patients with chronic hepatitis C virus (HCV) infection are often asymptomatic unless they have advanced liver disease. They usually have mild elevation of the serum ALT level, and about one third have persistently normal liver enzyme levels. Accordingly, as mentioned above, lack of sensitivity and specificity limit the use of liver function tests. For example, in some clinical conditions (e.g., cirrhosis), patients may have serum aminotransferase levels in the normal to near-normal range. In addition, several nonhepatic factors (Moseley, R. H. 1996) can affect the results of tests that measure specific hepatic function, such as serum albumin, total bilirubin, and prothrombin time (PT).
Several markers have been proposed for cirrhosis and for pre-cirrhotic fibrosis, for example the serum levels of aminoterminal propeptide of procollagen type III (PIIINP) or the aminoterminal domain of procollagen type IV (PIVNP). However, abnormal serum levels may also be observed in non-hepatic diseases. In addition, these markers too are not very accurate, since in the case of PIVNP, about 40% of patients with cirrhosis and about 55% of patients with severe fibrosis show normal PIVNP levels (2).
The only reliable and definitive test for liver function and status is a biopsy. However, biopsies cannot be used in standard tests, or for patients with mild conditions or even for routine periodic analysis in patients with severe liver disease.
The human asialoglycoprotein receptor (ASGPR) is expressed only in hepatocytes and serves in the clearance of asialoglycoproteins from the plasma (3). ASGPR levels are much lower in developing liver than in fully developed liver. The receptor level is also reduced in patients with cirrhosis and dramatically down-regulated in hepatocarcinomas (4).
The ASGPR is constructed of two subunits of related amino acid sequence, H1 (46 kD) and H2 (50 kD). H2a and H2b are two alternatively spliced variants of the ASGPR H2 subunit (5). H2a differs from H2b only by the presence of an extra pentapeptide in the exoplasmic domain next to the membrane-spanning segment (5). It was shown that H2a is rapidly cleaved next to this pentapeptide to a 35 kDa fragment, comprising the entire ectodomain, which is secreted, constituting a soluble form of the receptor (sH2a) (6). Membrane-bound H2a does not participate in a receptor complex with H1 as is the case for H2b, and thus it is not a subunit of the receptor but a precursor for the soluble secreted form.
Although H2a is a type II transmembrane protein, indirect evidence suggests that signal peptidase is probably responsible for the cleavage to the soluble form. ASGPR sH2a was found to be efficiently secreted from the human hepatoma cell line HepG2 (6). It was discovered that when H2a is expressed in stably transfected NIH 3T3 cells it is also cleaved, however only about 30% of sH2a can be Golgi processed and secreted from transfected fibroblasts and the rest is degraded at the ER (6).